STUDI DI ESPRESSIONE DI MICRORNA NELLE NEOPLASIE EMATOLOGICHE DEL CANE

Recent studies have identified a new class of short noncoding RNAs called microRNAs (miRNA) that are involved in gene expression negative regulation. Until now miRNAs were discovered in a several organisms, such as viruses, protozoa, plants, arthropods, fish, amphibians and mammals. The MicroRNAs constitute about 1-3% of predicted genes in animals genomes, and each miRNA can control hundreds of gene targets. A large number of studies in human medicine have suggested that miRNAs play important roles in innate immune response modulation and they are involved in normal hematopoiesis and haematological malignancies; miRNAs are differentially expressed in hematopoietic lineages and any alteration of these processes is directly associated with a dysregulation of miRNAs. In human the expression profiling of miRNAs serve as biomarkers for hematopoietic tumor classification, diagnosis and prognosis. The frequency and nature of some of these tumors are comparable in dogs and humans, it is possible to hypothesize that the analysis of microRNAs may play an important role also in canine hemato-oncology. The aim of this work was the investigation of the expression profile of a panel of putative miRNAs in dogs with different types of spontaneous lymphoid neoplasms (T-CLL, B-CLL, T Cells Lymphomas, B Cells Lymphomas, Splenic lymphomas). Often to validate a molecular marker in the dog it is difficult to have a sufficient number of clinical samples, for this reason it is necessary to make retrospective studies based on analysis that put to use archival samples, such as tissue formalin-fixed paraffin-embedded (FFPE) or stained smears. It has been reported that miRNA expression profile in FFPE samples correlates well with the corresponding fresh-frozen samples (Xi 2007), on the contrary for the cytological sample smears there is not any methods for the miRNA extraction and analysis. In this work the investigations were conducted both on fresh-frozen and retrospective samples like corresponding FFPE samples and stained smears on which we have standardized a procedure of extraction and analysis of microRNAs. According to the information obtained from literature or from databases, we have selected a panel of putative microRNAs known as ubiquitous or typical of human hematopoietic cells and with conserved sequences in canine genome. The expression levels of the miRNAs selected was determined through real time reverse transcription quantitative PCR (RT-qPCR) in lymphoid tissues of the dog, both healthy and affected by different spontaneous hematopoietic tumors, using TaqMan assays originally designed for the corresponding human microRNAs. Expression data were normalized against the most stable reference miRNAs ranked within a panel of candidate endogenous control (EC) genes through NormFinder and geNorm analysis. The results of this study confirm that also in dogs, as already demonstrated in humans, there is a differential expression of some miRNAs in various hematopoietic malignancies, this suggest the potential utility of miRNAs as a new diagnostic biomarker in the canine hemato-oncology. Moreover with this work makes available a procedure for analysis of microRNAs from cytological slides that allows to obtain comparable data to those obtained from fresh samples, this could be useful for retrospective studies on canine lymphoproliferative disorders, such as lymphoma and leukaemia, where stained smears are often the only archive material available.